Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway
Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway
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DOI：
10.1016/j.bpj.2016.03.001
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发表时间：
2016-04-26
影响因子：
3.4
通讯作者：
Falke, Joseph J.
中科院分区：
文献类型：
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作者：
Ziemba, Brian P.;Burke, John E.;Falke, Joseph J.
在趋化阿米巴细胞中，质膜的细胞质小叶上形成复杂的前缘信号传导电路，并指导肌动蛋白和膜重塑以推动前缘向上吸引剂梯度。该前沿电路包括一个假定的放大模块，其中 Ca2+-蛋白激酶C(Ca2+-PKC) 被假设磷酸化肉豆蔻酰化富含丙氨酸的 C 激酶底物 (MARCKS) 并释放磷脂酰肌醇-4,5-二磷酸 (PIP2)，从而刺激脂质激酶产生信号脂质磷脂酰肌醇-3,4,5-三磷酸 (PIP3)磷酸肌醇-3-激酶 (PI3K)。我们研究了这种假设的 Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 扩增模块，并使用单分子荧光测量其蛋白质成分的表面密度和活性来测试其关键预测。我们的研究结果表明，Ca2+-PKC 和 MARCKS 的 PIP2 结合肽一起调节游离 PIP2 的水平，而游离 PIP2 既可作为 PI3K 的对接靶标，又可作为底物脂质。在扩增模块关闭状态下，MARCKS 肽隔离 PIP2，从而抑制 PI3K 与膜的结合。在开启状态下，MARCKS 肽的 Ca2+-PKC 磷酸化可逆转 PIP2 隔离，从而释放多个 PIP2 分子，将多个活性 PI3K 分子招募到膜表面。这些发现 1) 显示 Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 系统在体外充当激活模块，2) 揭示激活的分子机制，3) 与现有的体内数据一致，4) 产生可在活细胞中测试的额外预测。更广泛地说，Ca2+-PKC 刺激的游离 PIP2 释放可以很好地调节其他 PIP2 结合蛋白的膜关联，这些发现说明了单分子分析在阐明膜表面多蛋白信号传导途径的关键动态和机制特征方面的能力。
In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca2+-protein kinaseC(Ca2+-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca2+-PKC and the PIP2-binding peptide of MARCKS modulate the level of free PIP2, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP2 and thereby inhibits PI3K binding to the membrane. In the on state, Ca2+-PKC phosphorylation of the MARCKS peptide reverses the PIP2 sequestration, thereby releasing multiple PIP2 molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca2+-PKC-stimulated release of free PIP2 may well regulate the membrane association of other PIP2-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.